The identification of immune responses that protect against mucosal invasion is of paramount importance in the development of a vaccine capable of blocking the sexual transmission of HIV. To this end, we have examined immune responses induced in gut-associated tissues in the macaque in response to transient infection with 2 molecular infectious clones of SIV and have correlated these responses with protection against intracolonic challenge with a high dose of the primary heterologous isolate, SIV/DeltaB670. Only 1 of 7 inoculated monkeys developed a disseminated infection. Of the remaining 6 animals, a transient infection was apparently induced in two, because, despite the inability to detect SIV or virus-specific immune responses in the peripheral blood, a high level of MHC class I restricted cytotoxic T lymphocyte (CTL) activity directed against viral env determinants was identified in the intestinal lamina propria. Six months post-exposure, these 7 animals and 4 naive controls were challenged intracolonically with SIV/DeltaB670. SIV/DeltaB670 sequences were successfully identified in peripheral blood mononuclear cells by 3 weeks postchallenge in all monkeys except the two monkeys with gut-associated CTL. The association of env-specific CTL with protection was further supported by its detection at 3 weeks postchallenge in the lamina propria of only those animals that were protected. These experiments provide the first direct evidence for a role for MHC class I restricted CTL in protection against mucosal exposure.